In a focal model of brain ischemia in the spontaneously hypertensive rat (SHR), microvascular perfusion, accumulation of inflammatory mediators, and indices of cellular injury have been correlated spatially and temporally by means of multiple label immunohistochemistry in order to study their role in acute stroke. The findings clearly demonstrate that progressive impairment of microcirculation occurs in regions of brain that undergo progressive damage in the early hours of ischemia. Inhibition of tumor necrosis factor-alpha (TNF-alpha) with TNF-binding protein reduces brain infarct volume in middle cerebral artery occlusion (MCAO) models in the rat and mouse. In addition, TNF-binding protein attenuates the progressive impairment of microvascular perfusion that occurs during the early hours of focal brain ischemia. These findings implicate TNF-alpha as a mediator of progressive brain damage during acute stroke. Lipopolysaccharide (LPS) pretreatment has been demonstrated to induce tolerance to focal brain ischemia in the MCAO model in the SHR. TNF binding protein blocks this tolerance implicating TNF-alpha as the mediator. This form of tolerance also reduces the degree of microcirculatory perfusion impairment in brain. Preconditioning with TNF by intracisternal injection of TNF induces tolerance to ischemia in the Balb/C mouse. In vitro models comprising various cellular elements of brain have been established in order to identify the mechanisms involved in the observed in vivo tolerance to ischemia of the brain pretreated either by tumor necrosis factor-alpha (TNF-alpha) or by hypoxia/ ischemia. TNF-alpha pretreatment of rat cortical astrocytes and brain microvascular endothelial cells (BMEC) renders these cells unresponsive to TNF activation 24 hours later in terms of up-regulation and expression of ICAM-1 (analyzed by FACS fluorescent cell ELISA as well as by Northern blot analysis of ICAM-1 mRNA). Ceramide is a lipid messenger implicated in TNF signaling. Addition of C-2 ceramide 30 min before or even 1 hour after TNF treatment resulted in inhibition of up- regulation of ICAM-1 at both protein and mRNA levels. HPLC determination of endogenous ceramide concentrations in TNF-activated cortical astrocytes and BMEC demonstrated two peaks of ceramide release: an early 1.9-fold increase at 15-20 minutes, and a late 2.5- fold increase at 18-21 hours after TNF addition that was associated with tolerance. Inhibition of ceramide synthase with Fumonisin B1 attenuated TNF-induced preconditioning of astrocytes in a dose- dependent manner indicating that late ceramide release is a mediator of TNF-induced tolerance. Primary cultures of neonatal cortical neurons were subjected to 60 minutes of hypoxia and reoxygenated for various times. Cell death was quantitated using the ethidium iodide exclusion fluorescence method. The number of dead cells gradually increased over 8 hours of reoxygenation reaching 21.9% at 8 hours of hypoxia. Pretreatment of neuronal cultures with short hypoxia (15 minutes) 24 hours prior to 60 minutes of hypoxia, protected neurons against hypoxia (number of dead cells was 9.4% versus 35% in non-pretreated cultures). Pretreatment with TNF (50 ng/ml) or the downstream signaling molecule,ceramide, (10 micromolar) also protected cortical neurons against 60 minutes of hypoxia. TNF-preconditioning can induce tolerance to subsequent TNF and hypoxia exposure in BMEC, astrocytes and cortical neurons. Ceramide appears to be involved in the intracellular signaling pathways leading to tolerance of brain cells to ischemia. - stroke model, cerebral ischemia, leukocytosis, microcirculation, reperfusion, cytokines, TNF alpha, ceramide